Cells die for many reasons, including normal physiological processes that control cell numbers in all tissues. Cell death can occur via natural physiological processes (the best known being apoptosis) as well as through accidental damage (necrosis)—caused in cells in culture, for example, by shear stress in culture vessels. In our bodies, dead cells are efficiently removed by phagocytic cells such as macrophages, otherwise they can cause tissue damage and can contribute to the development of disease. If they remain uncleared (as generally occurs in cell cultures), apoptotic cells progress to become necrotic (sometimes referred to as ‘secondary necrosis’). This progression is characterised by loss of plasma membrane integrity.
Current assays of cell viability are many and varied, simple and complex. For example, the simplest assays, such as vital dye exclusion, assess the ability of dyes that are excluded from viable cells to enter dead cells. Commonly-used examples of such dyes are trypan blue, propidium iodide and ethidium bromide. More complex assays include release of macromolecules, such as the enzyme lactate dehydrogenase (LDH), whose activity can be assessed via its activity on a suitable chromogenic substrate. These assays, however, are restricted to detection of the necrotic stage of cell death since both require loss of plasma membrane integrity for positivity. Full appreciation of the true viability of a population at a given time requires assessment of both dying (apoptotic) and dead (necrotic) cells. Full appreciation of the true viability of a population at a given time requires assessment of both dying (apoptotic) and dead (necrotic) cells. Assays of dying, apoptotic, cells are generally complex, requiring multiple steps (for example TUNEL-type assays and assays of caspase activation) and this can lead to technical problems such as false-positives. A common method makes use of the early changes in plasma membrane phospholipid asymmetry that are a hallmark of apoptosis. The phospholipid-binding protein, annexin V, under relatively high concentrations of extracellular Ca2+, binds to the phospholipid, phosphatidylserine (PS), that is rapidly exposed on the surface of apoptotic cells prior to loss of plasma membrane integrity. Antibody-mediated detection of PS, such as via Immunosolv's imab6 monoclonal antibody, obviate the need for extracellular Ca2+. There are currently a limited range of methods and kits available for achieving the selective removal of dying/dead cells from cell cultures. These include, for example, Miltenyi kits and imab6-coupled Dead-Cert™Nanoparticles produced by Immunosolv.
Despite the availability of these reagents and kits, there is a need for new improved techniques as although dyes like trypan blue can be used to accurately detect or label dead cells, it is not capable of highlighting dying cells. Furthermore, annexin V is Ca2+-dependent and thus inconvenient for measurement under conditions of low Ca2+ as might occur in suspension cultures commonly used in biomanufacturing.
In addition to the above, there is a need for additional methods of manipulating non-viable cells, improving the viability of cell cultures and the storage and transport of cells. Improved protocols for cell line establishment and productivity are also needed. Any method which lowers background activity due to dead cells and improves transfection efficiency would also represent a significant improvement over the prior art.
There is also a need for methods which manipulate cells at late stages of apoptosis as early apoptotic cells, which produce growth factors such as lactoferrin, can have positive effects on their surroundings [Bournazou, 2008 #3517].